A Recombinant Protein PCXZ And DNA Vaccine PDNA-PCXZ From Hepatitis C Virus Possess The Immunogenicity In BALB/c Mice

Hepatitis C virus is a positive RNA virus and a hepatotropic pathogen. The great genetic variability, which is the main reason for virus easy to escape from the host immune responses, is a major challenge for vaccine development. Based on the current knowledge of the mechanisms involved in control of viral infection, a vaccine should induce a multispecific and vigorous cellular host response, implicating both CD4+ and CD8+ T cells and a strong and cross-neutralizing antibody response.Combining a multiepitope antigen gene pcx in previous studies, three additional conserved B/T cell epitopes were selected to form a multiepitope gene pcxz in this study. The antigenic specificity of recombination PCXZ was determined by recognizing antibodies in serum samples from hepatitis C virus patients but not from healthy subjects or subjects who had the hepatitis B virus. The characteristics of PCXZ immunogenicity were evaluated in BALB/c mice. Strong antibody responses were generated in mice immunized with either naked PCXZ or PCXZ in Freund’s adjuvant. As to the T cells responses, Freund’s adjuvant significantly increased gamma interferon secretion and enhanced the lytic activity of cytotoxic T lymphocytes. The epitope Pa, one component of PCXZ, made the most significant contribution to specific CTL lysis; this epitope was also a B cells epitope and was able to induce high IgG titers.Considering the excellent character on inducing both humoral and cellular immune responses of plasmid DNA (pDNA) vaccine, the pDNA vaccine based on the multiepitope gene pcxz was developed and named cPCXZ. And mPCXZ which could express protein PCXZ binding the cellular membrane was constructed by inserting a murine IgG k-chain leader sequence and a platelet-derived growth factor receptor (PDGFR) transmembrane sequence linked to the upstream and downstream of pcxz, respectively. The PCXZ was successfully and correctly expressed in cells transfection with cPCXZ and mPCXZ, respectively, and recognized by the HCV positive sera.BALB/c mice was vaccinated with pDNA cPCXZ or mPCXZ directly or co-delivery of molecular adjuvants pGM-CSF. An effectively specific CD4+ and CD8+ T cells responses were induced in all immunized mice, especially in mPCXZ and pGM-CSF co-administrated mice, indicating that mPCXZ was better than cPCXZ, and pGM-CSF enhanced the cellular-mediated immune response. To improve the B cells immune response, the recombination PCXZ was boosted after twice pDNA primes in mice, and a high titer of specific IgG antibody was induced with no significant difference between cPCXZ and mPCXZ immunization groups. As to the epitope Pa, CD4+ and CD8+ T cells responses were elicited significantly, moreover, the specific IgG antibody was induced; while for the epitopes of Pb and Pc, only the CD8+ T cells response was significant. In summary, in this study, the mPCXZ pDNA vaccine with the membrane-archored PCXZ expression, which was constructed from several conserved epitopes of HCV, was able to elicit strong T cell immune response in BALB/c co-administrated with molecular adjuvant pGM-CSF. And the recombinant PCXZ was then boosted. Finally a high titer specific IgG was induced. These provide a basis for further research on HCV infection model, and also illustrate the potency of this pDNA vaccine and immunization pattern in the field of HCV vaccine development.